Ataxia, Leigh syndrome, retinitis pigmentosa, muscle weakness and familial bilateral striatal necrosis can result from damage to the genes that encode FoF1 ATP synthase subunits. The FoF1 ATP synthase has two opposed rotary molecular motors connected by a common axle. The integral membrane Fo motor uses proton- motive force (PMF) to drive axle rotation for F1-dependent ATP synthesis. In vivo, FoF1 maintains the [ATP]/[ADP][Pi] ratio far from equilibrium, enabling high [ATP] to provide an energy source for cellular processes. The Fo motor uses a Brownian ratchet to bias clockwise rotation against an F1 motor-imposed load. We recently observed a previously unknown interaction between Fo subunits a and c of FoF1 when ATPase-driven rotation is slowed by a viscosity-induced load. A striking feature of this interaction is that it forms a tether that limits rotation to 360. The cD44N/cR50 mutant eliminates tether formation and causes loss of oxidative phosphorylation-dependent E. coli growth, indicating that the tether is an important Fo motor component for ATP synthesis in vivo. A mechanistic hypothesis is proposed where the tether enables the Fo motor to ratchet clockwise rotation against an F1 motor-imposed load during ATP synthesis. The focus of the work proposed here is to test this Fo motor mechanism hypothesis when FoF1 synthesizes ATP. The Fo mechanism is poorly understood compared to that of F1, in part, because of membrane-associated technical problems that make it very difficult to carry out single-molecule studies on Fo. A novel assembly of supported planar lipid bilayers containing oriented FoF1 ATP synthase molecules on a microscope slide will now be made to enable single molecule rotation measurements during ATP synthesis with a time resolution to 5 s at unprecedented signal-to-noise. The specific aims of the project will: (1) determine rotational velocity and torque generated by the Fo motor during ATP synthesis as a function of PMF; (2) determine tether formation and duration during ATP synthesis as a function of a load on Fo imposed via viscosity, by increasing ATP/ADP.Pi, by decreasing Fo driving force relative to the F1 load, and by mutant analysis; (3) identify the 9 ms catalytic dwell during ATP synthesis, and determine if the other 9ms dwell is substrate-waiting; and (4) test the escapement mechanism hypothesis for coupling rotation with ATP synthesis through mutant analysis. Our discovery of the previously unknown tether between subunit a and subunit-c residues cD44 and cR50 provides a new window with which to examine the mechanism by which the Fo motor powers rotation to catalyze ATP synthesis. Through the use of our innovative approach for the assembly of supported planar lipid bilayers in combination with our novel nanorod assay, the experiments proposed here will provide important new insight concerning several fundamental aspects of the mechanism of the Fo molecular motor, and the means by which it interfaces with the F1 motor to catalyze the synthesis of ATP.